The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties

The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions

“…These findings were supported by our observations for rPmpD under non-reducing and reducing SDS PAGE, which indicate that even more extensive covalent disulphide bonding leads to formation of oligomeric complexes. Our data expand upon recent findings involving the 23 kDa Chlamydia pneumoniae Pmp21 passenger domain fragment, where a different mechanism of oligomeric interaction involving FxxN repeat motifs was proposed [ 18 ]. Indeed, we have observed that non-covalent interactions may also contribute to assembly of of rPmpD oligomers, evidenced by the dissociation of some monomeric protein in non-reducing SDS PAGE, although it is apparent that disulphide bonding is yet another important mechanism by which these cysteine-rich T5SS proteins interact, and it is likely that both types of interaction may occur in multiple chlamydial species.…”

“…We provide a biophysical characterization of rPmpD, identifying for the first time, a unique role for disulphide bonds in oligomerization and self-interaction of this T5SS protein, as well as in the preservation of monomeric secondary structure, showing that all 18 cysteine residues are disulphide-bonded in the dominant proteoforms. Circular dichroism analysis shows that rPmpD consists primarily of β–sheet secondary structure, which is consistent with biophysical analyses presented for the truncated 23 kDa Pmp21 domain in Chlamydia pneumoniae , as well as experimentally solved crystal structures of other Gram-negative bacterial T5SSs [ 18 , 22 ]. Finally, we show that rPmpD possesses adhesin-like characteristics, and that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate adhesion of rPmpD-coated beads to the mammalian Hak cell line, highlighting a putative role for neutralizing anti-rPmpD antibodies in in vitro studies, which is consistent with our previous observations in vivo [ 8 ].…”

“…In contrast to the Ct MOMP that is highly hydrophobic and poorly soluble in the absence of detergents and lipid bilayers [ 24 ], both monomeric and oligomeric rPmpD were found to partition almost exclusively in to the aqueous rather than the detergent phase during Triton X-114 phase separation of LPS ( Fig 1C ), showing predominantly hydrophilic properties. Interestingly, the elution profile of rPmpD closely resembles that of the Pmp21 23 kDa domain previously described [ 18 ], and we next sought to determine the biophysical characteristics of the purified Ct rPmpD 65 kDa passenger domain fragment, as well as delineate the biochemical mechanisms of Ct PmpD interaction and native secondary structure.…”

“…Spectra for oligomeric (Peak ‘O’) and monomeric (Peak ‘M’) rPmpD fractions are indicative of predominantly β-sheet secondary structure interspersed with random coil elements, similar to the Pmp21 ortholog in C . pneumoniae [ 18 ].…”

“…An accumulating body of evidence in Chlamydia pneumoniae is now beginning to inform the molecular interactions between Pmp21 (the ortholog of PmpD) and mammalian cells, and although no host cell ligand has been defined for any Ct Pmp, it has been shown that these outer membrane proteins function as adhesins. It was first observed that FxxN and GGA(I,L,V) repeat motifs influence host cell adhesion and self-interaction, and subsequently, that mutating one of these repeat motifs in a truncated 23 kDa C-terminal recombinant Pmp21 fragment results in reduced ability to form oligomers [ 5 , 18 , 19 ]. PmpD contains 17 GGA(I,L,V) and 19 FxxN tetrapeptide sequences that may indeed play similar roles in Ct , as these repeat motifs are a characteristic feature of chlamydial autotransporter passenger domains, and markedly over-represented in Pmps relative to the rest of the proteome [ 20 ].…”